Project Summary/Abstract The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a key regulator of cell growth and proliferation that integrates mitogen cues, energy stress, and nutrient availability to coordinate central anabolic and catabolic cellular processes1-4. Aberrant mTORC1 signaling has been implicated in a variety of human diseases including cancer, diabetes, and epilepsy as well as normal physiological processes such as aging1,5-6. Accordingly, the molecular basis for mTORC1 activation is of significant fundamental and pathophysiological interest. Coincident detection of growth factor signals and nutrients, including the amino acids leucine and arginine, is required for mTORC1 activation1-4. Availability of these amino acids is conveyed to mTORC1 via the heterodimeric Rag GTPases, which recruit mTORC1 to the lysosomal surface for activation by the mTOR kinase activator Rheb11-12. The GATOR1 complex inhibits mTORC1 recruitment through its function as a GAP toward RagA/B. The GATOR2 complex interacts with and inhibits GATOR118. In the absence of amino acids, the leucine sensor Sestrin2 and arginine sensor CASTOR1 bind to and inhibit GATOR2 thereby repressing mTORC1 activation19-24. Despite identification of these components of the amino acid sensing machinery and their respective functions, the mechanism by which the GATOR2 complex inhibits GATOR1 and leads to mTORC1 activation remains elusive. As the node that integrates the availability of leucine and arginine, GATOR2 is a key component of the nutrient sensing branch upstream of mTORC1. Our preliminary results suggest that GATOR2 functions as an E3 ubiquitin ligase and that this activity is essential for mTORC1 to sense amino acids. Elucidation of the molecular mechanism of GATOR2 will provide key insight into how mammalian cells sense and respond to essential nutrients and may also provide novel targets for therapeutic agents designed to ameliorate deregulated mTORC1 signaling. To characterize the molecular function of GATOR2, we propose the following aims: 1. Characterize the E3 ubiquitin ligase activity of GATOR2 2. Identify substrates of GATOR2 3. Determine how GATOR2 ubiquitin ligase activity regulates mTORC1 activation in vivo